Legionella pneumophila, the causative organism of Legionnaires'disease, is a fresh water bacterium that can invade amoebae and macrophages and replicate in membrane bound phagosomes. These virulence phenotypes require a Type IV secretion system (T4SS), currently proposed to be the T4SS encoded by the dot/icm loci. The requirement for the Dot/lcm T4SS is based on defects in internalization, delay of phagosome acidification and intracellular replication phenotypes when amoebae and macrophages are infected with broth stationary cultures of Legionella dot/icm null mutants. The Pi's laboratory showed (i) that when broth cultures of dotA and dotB null mutants are incubated in water prior to infection, defective entry and defective phagosome acidification were reversed and levels of the parental strain JR32 achieved and (ii) after encystment in amoebae, dotA and dotB mutants were able to replicate intracellularly in amoebae trophozoites. These data suggest an alternative T4SS is involved in reversal of defective phenotypes in dotA/B mutants following exposure to water or encystment, mimics of environmental niches in the spread of Legionnaires'disease. The Pi's laboratory implicated the T4SS encoded by the Legionella Ivh locus in reversal of defective virulence phenotypes in dotA/B mutants. The Lvh T4SS locus is currently thought to be dispensable for L pneumophila virulence phenotypes. Since the Dot/lcm T4SS is a major focus in Legionella research, implication of the Lvh T4SS in virulence phenotypes identifies a new mechanism in Legionella pathogenesis. The Pi's laboratory implicated two tetratricopeptide repeat (TPR)-containing proteins in entry and phagosome acidification phenotypes of strain JR32 and in response to water treatment. The Specific Aims of this proposal are to (1) define the function of the Lvh T4SS and its component proteins in Legionella virulence phenotypes, including translocation of known Dot/lcm T4SS effector proteins, alteration of phagosome trafficking and intracellular multiplication in amoebae and macrophage host cells, (2) identify effector proteins of the Lvh T4SS and test their role in L. pneumophila virulence phenotypes and (3) define the virulence roles of the TPR-containing proteins and identify which Legionella proteins interact with those TPR-containing proteins. The proposed research will be significant in defining the role of a second T4SS and Legionella TPR-containing proteins in virulence phenotypes. The Pi's research (i) demonstrates that mimics of environmental niches can affect virulence mechanisms of L. pneumophila and perhaps other pathogens of environmental origin and (ii) suggests that thwarting the bacterial response to environmental cues that mediate transition from free living organism to human pathogen may be a promising approach for control of Legionnaires'disease.